The basic goal of the research proposal outlined here is the understanding of the biochemical mechanisms underlying gene expression in Escherichia coli. A major effort of this laboratory has thus been directed toward the elucidation of the enzymatic basis for the synthesis of RNA and protein. The specific aims of the research proposed are directed toward the elucidation of the following major areas of interest: (1) Further characterization of DNA-dependent RNA polymerases from E. coli and T3 bacteriophage infected E. coli cells will be carried out. Particular attention will be focused on control mechanisms operating at the level of initiation and termination of RNA synthesis by these polymerases from phage T3 DNA template. The oligonucleotide sequences on DNA template that determine initiation and termination specificity of these two polymerases will be determined. In addition, complete biochemical characterization of T3 polymerase particularly with regard to characterization of multiple active sites involved in the various discrete steps of the polymerase reaction will be carried out. (2) Further studies on the mechanism of initiation of protein synthesis directed by 'natural' mRNA polymerase products will be carried out. Studies will be carried out to further elucidate the role of each of the initiation protein factors - IFl, IF2 and IF3 - in initiation complex formation. Particular attention will be directed towards elucidation of the role of factor IF3 in mRNA selection process. In addition, the ribosomal sites involved in initiation complex formation will be characterized. (3) Biochemical characterization of mRNA degradation will be carried out. Since the RNA polymerase products (mRNA) contain the triphosphate moiety at the 5'-end (pppApXpY---) and degradation of mRNA is known to occur in vivo from the 5' yields 3' direction, it will also be of interest to determine the role of 5'-triphosphate termini in degradation of RNA polymerase products in vitro.